PROJECT SUMMARY/ABSTRACT Structural biology of retrotransposition. Non-long terminal repeat (non-LTR) retroelements are mobile genetic elements that are able to copy and paste themselves into new locations in DNA genomes using a reverse transcriptase and an RNA intermediate. These retroelements comprise at least 46% of the human genome and little is known about their precise mechanism of integration into double stranded DNA. LINE elements are a particularly abundant class of retroelements in the human genome and have large effects on gene expression through insertion near transcription promoters. In addition, aberrant retrotransposition by LINE elements can result in the development of cancer through disruption of tumor suppressor genes. Despite more than 30 years of study, there is no atomic model that would explain the mechanism of retrotransposition at the molecular level. This is due to the fact that LINE elements are difficult targets for structure determination because they exhibit very low levels of retrotransposition in vitro. Group II introns are thought to be the ancestors of eukaryotic non-LTR retroelements, such as the LINE elements found in humans. Group II retroelements are a more tractable model system for structure determination due to their stability and high activity of retrotransposition in vitro. To gain structural insight into retrotransposition, we aim to use single- particle cryo-EM to solve high-resolution structures of a group II intron retroelement. Specifically, we will obtain structures of the retroelement during the different stages of retrotransposition into dsDNA. We will use a combination of biochemical, genetic, and structural approaches to: 1) Determine the structure of a free group II intron retroelement. 2) Determine the mechanism of integration into a target double-stranded DNA. This is expected to have direct parallels with retrotransposition catalyzed by mammalian retroelements. We will capture different stages of retrotransposition to create a `molecular movie' of the entire process. This will represent the first atomic model of retrotransposition.